Identication of Novel Hub Genes Through Expression Proles Analysis in Adrenocortical Carcinoma Chenhe Yao Beijing Zhicheng Biomedical Technology Co,Ltd Xiaoling Zhao Beijing Zhicheng Biomedical Technology Co,Ltd Xuemeng Shang Beijing Zhicheng Biomedical Technology Co,Ltd Binghan Jia Beijing Zhicheng Biomedical Technology Co,Ltd Shuaijie Dou Beijing Zhicheng Biomedical Technology Co,Ltd Weiliang Ye Beijing Zhicheng Biomedical Technology Co,Ltd Yuemei Yang ( [email protected] ) Beijing Zhicheng Biomedical Technology,Co.,Ltd. Research Keywords: Adrenocortical carcinoma, biomarkers, gene expression proling, Co-expression, prognosis Posted Date: July 1st, 2020 DOI: https://doi.org/10.21203/rs.3.rs-38768/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/21 Abstract Background: Adrenocortical carcinoma (ACC) is a heterogeneous and rare malignant tumor associated with a poor prognosis. The molecular mechanisms of ACC remain elusive and more accurate biomarkers for the prediction of prognosis are needed. Methods: In this study, integrative proling analyses were performed to identify novel hub genes in ACC to provide promising targets for future investigation. Three gene expression proling datasets in the GEO database were used for the identication of overlapped differentially expressed genes (DEGs) following the criteria of adj.P.Value<0.05 and |log2 FC|>0.5 in ACC. Novel hub genes were screened out following a series of processes: the retrieval of DEGs with no known associations with ACC on Pubmed, then the cross-validation of expression values and signicant associations with overall survival in the GEPIA2 and starBase databases, and nally the prediction of gene-tumor association in the GeneCards database. Results: Four novel hub genes were identied and two of them, TPX2 and RACGAP1, were positively correlated with the staging. Interestingly, co-expression analysis revealed that the association between TPX2 and RACGAP1 was the strongest and that the expression of HOXA5 was almost completely independent of that of RACGAP1 and TPX2. Furthermore, the PPI network consisting of four novel genes and seed genes in ACC revealed that HOXA5, TPX2, and RACGAP1 were all associated with TP53. Conclusions: This study identied four novel hub genes (TPX2, RACHAP1, HXOA5 and FMO2) that may play crucial roles in the tumorigenesis and the prediction of prognosis of ACC. Introduction Regarded as an orphan disease with an annual incidence worldwide between 0.7 and 2 cases/million, adrenocortical carcinoma (ACC) is a heterogeneous malignancy that originates from the adrenal cortex [1–3]. Even though it can occur at any age group, ACC displays a bimodal incidence pattern, peaking at adults, especially women, aged 40–50, and children aged 1–5 [4, 5]. In addition to the common and nonspecic attributes of abdominal mass effect, endocrine disturbances can be seen in 40–60% of adult patients and up to 90% in children with ACC [6–8]. At present, authenticated prognostic markers of ACC include initial staging, resection margin status, Ki-67 staining state, autonomous cortisol secretion, and the general condition of patients [9]. Even though these markers serve as relatively good prognostic tools, the outcome of ACC remains rather variable owing to its nature of heterogeneity even within the same disease stage or the same resection margin status [10]. Importantly, the initial staging of ACC has become the foremost factor in the determination of prognosis, with a ve-year survival of 81% for stage I and of only 13% for stage IV [3, 6, 11]. Unfortunately, early diagnosis of ACC serves as a vast roadblock due to the lack of specic symptoms and the rarity of this malignancy [10, 12]. Currently, limited treatment options are another Gordian knot in ACC. While surgical resection is the last straw of curative treatment for ACC, recurrence, and progression are common events, even in patients undergoing R0 resection [11]. Adjuvant therapies, including the only formally approved drug Mitotane and radiotherapy, Page 2/21 have shown certain improvements on the recurrence-free survival in ACC, but not on the overall survival [3, 13–15]. Even though the management of ACC remains challenging because of the heterogeneity and unpredictability, the advances in gene analysis with the help of bioinformatics have beneted the identication of promising targets and established novel approaches for research, to improve our understanding of ACC [5, 11]. Therefore, we aimed to identify novel hub genes that may play essential roles in ACC using public datasets to elucidate the development and the prediction of prognosis of ACC in this study. An integrated analysis was conducted using the comprehensive gene expression data to distinguish the differentially expressed genes (DEGs) in ACC. Novel hub genes were screened considering the gene expression and the relation to overall survival using multiple databases. Gene expression analysis and survival analysis were used to screen novel hub genes. Co-expression analyses and the construction of protein-protein interaction (PPI) network consisting of hub genes and seed genes in ACC were completed to offer new opportunities for the investigation of possible mechanisms underlying ACC and thus facilitate the stratication and the prediction of prognosis for patients with ACC. Materials And Methods Inclusion of gene expression datasets. The Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) was utilized for the searching of gene expression proling datasets to identify the DEGs comparing ACC with normal adrenal gland. Keyword: ‘adrenocortical carcinoma’. Inclusion criteria: 1. Organism: Homo sapiens; 2. Experiment type: Expression proling by array; 3. Contains both ACC tissues and normal adrenal tissues (NT). GSE90713 (number of specimens, NT:ACC = 5:57), GSE19776 (NT:ACC = 4:59), and GSE14922 (NT:ACC = 4:4) met all the inclusion criteria and were enrolled in this study. Identication and representation of DEGs. To determine the DEGs, the web tool GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/) was used. Through Benjamini & Hochberg false discovery rate method, the adjusted P-value (adj.P.Value) was selected to provide an appropriate balance between the discovery of statistically signicant genes, and the limitation of false positives. DEGs (adj.P.Value < 0.05 and |log2 FC|>0.5) comparing ACC with NT from the three datasets were retrieved. Volcano plots were performed to represent the distribution of genes between ACC and NT for three datasets respectively. Overlapped DEGs were then identied using Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/index.html) for further analysis. The hierarchical clustering of overlapped DEGs in three datasets for visualization of expression patterns was performed in R. Screening of novel hub genes. To determine novel hub genes that might play crucial roles in the development or the prediction of prognosis in ACC, the following procedures were performed. Firstly, using queries for [“gene name of a Page 3/21 specic DEG” and “adrenocortical carcinoma”] on Pubmed to screen novel genes, with the exclusion criterion: retrieved results showing any known correlation between target genes and adrenocortical carcinoma. Secondly, the survival analysis by overall survival (OS) method and the expression values of each gene in ACC compared with NT were performed through GEPIA2 (http://gepia2.cancer- pku.cn/#index) and starBase (http://starbase.sysu.edu.cn/), with the inclusion criteria: 1) retrieved results were consistent in two databases; 2) retrieved results were statistically signicant as P < 0.05. Additionally, the GeneCards database (https://www.genecards.org/) was used to provide information concerning gene-disease associations, especially gene-tumor associations in this study, to help locate possible novel genes that may contribute to the development of ACC. PPI network construction. To understand the possible mechanisms underlying novel hub genes in ACC, we constructed a PPI network. Known crucial genes that contribute to the development of ACC were obtained using Phenolyzer software (http://phenolyzer.wglab.org) and were referred to as seed genes, using “adrenocortical carcinoma” as the disease term. Seed genes and identied novel hub genes were then entered into the STRING database (https://string-db.org/). Results were retrieved after choosing Homo sapiens as the organism under the default parameters and visualized in a PPI network, constructed in the Cytoscape software (http://www.cytoscape.org/). Data analysis. Three datasets used in this present study are all available in the GEO database. Expression values of genes in different stages of ACC were retrieved from the UALCAN database (http://ualcan.path.uab.edu/analysis.html). Co-expression analysis was performed using data from the starBase database. All survival analyses were carried out in the GEPIA2 database and represented by Kaplan-Meier curves, and p values between each group pair were analyzed by the log-rank test. Statistical signicance of different experimental data was analyzed using GraphPad Prism 8. P < 0.05 was considered to be statistically signicant. Results 38 DEGs were identied in ACC. Repeated measures and DEGs without gene symbols were excluded from the results. Based on the cutoff criteria, 267, 242, and 186 up-regulated DEGs comparing ACC with NT were retrieved in the GSE90173, GSE19776, and GSE14922 datasets, respectively; 714, 943 and 202 down-regulated